NMR studies of a tetrasaccharide from hyaluronic acid.
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Carbohydrate Conformation↗
Biomedical subjects
L Rodén
Publications and source records attributed to L Rodén.
Glucuronosyl transfer to galactose residues in the biosynthesis of HNK-1 antigens and xylose-containing glycosaminoglycans: one or two transferases?
An early step in the assembly of the xylose----serine-linked proteoglycans is the transfer of glucuronic acid to the C-3 position of a galactose residue in the carbohydrate-protein linkage region. Since a similar reaction occurs in the biosynthesis of NHK-1 antigens, the question arose whether these processes are catalyzed by the same enzyme. In the present study, the proteoglycan-related glucuronosyltransferase activity in embryonic chick brain was found to be firmly membrane-associated, while the majority of the activity towards N-acetyllactosamine - a model substrate for HNK-1 antigen biosynthesis - was readily solubilized. No activity towards N-acetyllactosamine was found in embryonic chick cartilage, which is a rich source of the proteoglycan-related enzyme. Together with the results of mixed substrate experiments, these findings strongly indicate the existence of two separate glucuronosyltransferases catalyzing transfer to galactose residues.
Animals↗
Biosynthesis of heparin. Enzymatic sulfation of pentasaccharides.
Heparin-derived pentasaccharides with the general structures GlcN-GlcA/IdoA-GlcN-GlcA/IdoA-GlcN (where GlcA represents D-glucuronic acid and IdoA represents L-iduronic acid) and GlcNSO3-GlcA/IdoA-GlcNSO3-GlcA/IdoA- GlcNSO3 (where -NSO3 represents an N-sulfate group) were tested as exogenous sulfate acceptors in incubations with adenosine 3'-phosphate 5'-[35S]phosphosulfate and microsomal enzymes from a heparin-producing mouse mastocytoma. No transfer occurred to the N-unsubstituted pentasaccharide containing only L-iduronic acid, but the other three isomers incorporated various amounts of 35S, which was totally present in N-sulfate groups. After complete chemical N-sulfation, all four pentasaccharides served as acceptors in O-sulfotransferase reactions and incorporated from 20 to greater than 200 times as much radioactivity as did the nonsulfated parent compounds. The C-6 position of the internal glucosamine unit was labeled preferentially, irrespective of the structures of the adjacent hexuronic acid units. Significant 2-O-35S-sulfation of IdoA units occurred in both -IdoA-Glc-NSO3-GlcA- and -GlcA-GlcNSO3-IdoA- sequences, whereas no significant sulfation of GlcA residues was detected. The pentasaccharide GlcNSO3-GlcA-Glc-NSO3-GlcA-GlcNSO3 thus can be used as a selective substrate in assays for glucosaminyl-6-O-sulfotransferase activity. The antithrombin-binding region, essential for the blood anticoagulant activity of heparin, has been identified as a pentasaccharide sequence with the predominant structure GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-++ +IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R represents either a sulfate or an acetyl group and -OSO3 represents an O-sulfate/ester sulfate group, with locations of O-sulfate groups indicated in parentheses) (Lindahl U., Thunberg, L., Bäckström, G., Riesenfeld, J., Nordling, K., and Björk, I. (1984) J. Biol. Chem. 259, 12368-12376). The products of [35S]sulfate transfer to the pentasaccharide GlcNSO3-GlcA-GlcNSO3-IdoA-GlcNSO3 contained molecules with high affinity for antithrombin, corresponding to 0.3-0.5% of the total label. Structural analysis suggested the occurrence of O-[35S]sulfate groups at both C-6 of the nonreducing terminal glucosamine unit and C-3 of the internal glucosamine unit. No products with high affinity for antithrombin were formed from the pentasaccharides that had a different monosaccharide sequence than the binding region; and moreover, these oligosaccharides appeared unable to incorporate glucosaminyl 3-O-sulfate groups. These findings point to the importance of the uronic acid sequence in the generation of the antithrombin-binding region of heparin.
Animals↗
Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans.
The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.
Computer Graphics↗
Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides.
A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.
Acetylglucosamine↗
N-acetylglucosamine-6-phosphate deacetylase in hepatocytes, Kupffer cells and sinusoidal endothelial cells from rat liver.
The activity of N-acetylglucosamine-6-phosphate deacetylase, a key enzyme in the pathway of N-acetylglucosamine catabolism, was measured in hepatocytes, Kupffer cells and sinusoidal endothelial cells from rat liver and cultured human skin fibroblasts. Kupffer cells and endothelial cells had similar high levels of deacetylase activity that were more than twice the level observed in fibroblasts. In contrast, hepatocytes had extremely low activity (several hundredfold less than Kupffer cells and endothelial cells). A major implication of deacetylase deficiency in hepatocytes is that N-acetylglucosamine generated as a result of the catabolism of complex carbohydrates in these cells cannot enter glycolysis and must be largely reused for the synthesis of plasma glycoproteins and other N-acetylglucosamine-containing macromolecules.
Amidohydrolases↗
A genetic defect in the biosynthesis of dermatan sulfate proteoglycan: galactosyltransferase I deficiency in fibroblasts from a patient with a progeroid syndrome.
A small proteoglycan that contains only a single dermatan sulfate chain is the main proteoglycan synthesized by skin fibroblasts. Fibroblasts from a patient with progeroidal appearance and symptoms of the Ehlers-Danlos syndrome have a reduced ability of converting the core protein of this proteoglycan into a mature glycosaminoglycan chain-bearing species. This abnormality is the consequence of a deficiency in galactosyltransferase I (xylosylprotein 4-beta-galactosyltransferase; EC 2.4.1.133), which catalyzes the second glycosyl transfer reaction in the assembly of the dermatan sulfate chain. The glycosaminoglycan-free core protein secreted by the patient's fibroblasts bears an unsubstituted xylose residue. The mutant enzyme is abnormally thermolabile. Preincubation of fibroblasts at 41 degrees C leads to a further reduction in the production of mature proteoglycan and affects the capacity for glycosaminoglycan synthesis on p-nitrophenyl beta-D-xyloside more strongly in the mutant than in control cells.
Carbohydrate Sequence↗
Enzymic pathways of hyaluronan catabolism.
The enzymic degradation of hyaluronan in mammalian tissues takes place in two phases, encompassing breakdown of the polysaccharide to its monosaccharide constituents and subsequent utilization of the monosaccharide products. Degradation to the monosaccharide components is effected by the concerted action of three enzymes, hyaluronidase, beta-D-glucuronidase and beta-N-acetyl-D-hexosaminidase. The relative contributions of hyaluronidase and the two exoglycosidases to the physiological catabolism of hyaluronan are not yet known but consideration of the kinetic properties of the three enzymes clearly indicates that hyaluronidase is best suited for the initial attack on the polysaccharide, inasmuch as its Km for hyaluronan is 1000- to 10,000-fold lower than that estimated for beta-D-glucuronidase. Recent investigations in the authors' laboratories have been focused on the catabolism of hyaluronan and other complex carbohydrates in liver, since the sinusoidal endothelial cells in this organ are the main sites for degradation of circulating hyaluronan. Assay of ten lysosomal hydrolases in isolated rat liver cells showed considerably higher activities in Kupffer cells and endothelial cells than in hepatocytes for nine of the enzymes, including beta-D-glucuronidase and beta-N-acetyl-D-hexosaminidase. The activity of N-acetylglucosamine-6-phosphate deacetylase, a key enzyme in the metabolism of the N-acetylglucosamine released by the lysosomal degradation of hyaluronan and other complex carbohydrates, has also been determined. High deacetylase activities were observed in both Kupffer cells and endothelial cells but, surprisingly, virtually no activity was detected in hepatocytes. This finding implies that N-acetylglucosamine cannot be degraded in hepatocytes and must be largely reutilized in the synthesis of new macromolecules. Further studies of the enzymes involved in hyaluronan degradation and N-acetylglucosamine utilization in the liver are under way.
Animals↗
Identification of oversulphated galactosaminoglycans in intestinal-mucosal mast cells of rats infected with the nematode worm Nippostrongylus brasiliensis.
The oversulphated galactosaminoglycans synthesized by rat mucosal mast cells were isolated from the small intestine of animals infected with the nematode Nippostrongylus brasiliensis, which causes proliferation of these cells. The 35S-labelled polysaccharides were degraded by digestion with chondroitinase ABC, and the structures of the disaccharide products were determined by cleavage with mercuric acetate followed by electrophoretic characterization of the resultant sulphated monosaccharides. It was concluded that about half of the disulphated disaccharide units in the polysaccharide consisted of chondroitin sulphate E-type structures [GlcA-GalNAc(4,6-di-OSO3)], in which both sulphate groups were located on the N-acetylgalactosamine unit. The remainder consisted of isomeric structures with one sulphate group on the N-acetylgalactosamine residue and one on the hexuronic acid unit and presumably represented the dermatan sulphate-type sequence [IdoA(2-OSO3)-GalNAc(4-OSO3)].
Animals↗
Synthesis of proteoglycans in rat mucosal keratinocytes.
The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [35S] sulfate or D-[1-3H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the 35S-labelled glycosaminoglycans showed that heparan sulfate was the major component (approximately 90%) and that small amounts (approximately 10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-3H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [35S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a Kav of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.
Animals↗
Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I.
We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.
Animals↗
Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose.
Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.
Cell Line↗
Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver.
A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.
Amidohydrolases↗
Proteoglycans and hypertension: III: Aorta proteoglycans in Dahl salt-sensitive hypertensive rats.
The vascular proteoglycans probably have an important influence on the biomechanical properties of blood vessels and, therefore, may play a role in the development or maintenance of hypertension. In the aorta of the spontaneously hypertensive rat, the authors previously observed an increased content of chondroitin sulfate, an increased incorporation of [35S]sulfate into proteoglycans, and qualitative alterations in the [35S]polysaccharides compared to the normotensive Wistar Kyoto rat. To determine if these differences were related to hypertension or to strain variations, normotensive and hypertensive Dahl S rats were studied. There was a significant elevation (70%) in the aorta content of chondroitin sulfate, whereas the dermatan sulfate and hyaluronic acid contents were similar in the two groups. The in vitro incorporation of [35S]sulfate was increased 2.6-fold in the hypertensive animals. No differences between the two groups were observed with respect to the gel chromatographic profiles of the [35S]proteoglycans or the charge density of the [35S]glycosaminoglycans, as assessed by ion exchange chromatography. It was concluded that the increase in chondroitin sulfate and [35S]sulfate incorporation into proteoglycans occurred as a result of hypertension, regardless of genetic factors.
Animals↗
Oligosaccharide substrates for heparin sulfamidase.
The Sanfilippo A syndrome is characterized by a deficiency in heparin sulfamidase, which removes the N-sulfate groups of heparan sulfate and heparin in the course of normal catabolism of these polysaccharides. [N-35S]Heparin is the most commonly used substrate for the assay of sulfamidase activity but has certain disadvantages which have prompted us to search for alternative substrates. We report here on the use of heparin oligosaccharides for this purpose. The trisaccharide, GlcN-IdoUA-GlcN, and the pentasaccharide, GlcN-GlcUA-GlcN-GlcUA-GlcN, were N-sulfated with [35S]sulfur trioxide-trimethylamine complex; the tetrasaccharide, GlcN-UA-GlcN-UA, and the pentasaccharide, GlcN-IdoUA-GlcN-IdoUA-GlcN, were labeled by reduction with sodium borotritide followed by chemical N-sulfation. When incubated with sonicates of cultured skin fibroblasts from normal individuals, all four oligosaccharides were found to serve as substrates for heparin sulfamidase. Fibroblast sonicates from patients with the Sanfilippo A syndrome had little or no activity toward these substrates. Optimal activity of the enzyme was at pH 4.4-4.5. Comparison of the kinetic parameters showed that heparin had a lower Km than the oligosaccharides, whereas the Vmax values of the latter were higher than for heparin.
Cells, Cultured↗
Proteoglycans and hypertension. I. A biochemical and ultrastructural study of aorta glycosaminoglycans in spontaneously hypertensive rats.
The extracellular matrix of blood vessel walls contains elastin, collagen, and proteoglycans, all of which can affect vascular resistance and, hence, blood pressure by virtue of their biomechanical properties. In the present study, we have begun to explore the possibility that proteoglycans may play a role in the pathophysiology of hypertension by analyzing, qualitatively and quantitatively, the polysaccharide components of proteoglycans from aorta of two normotensive rat strains, Wistar Kyoto (WKY) and Wistar rats, and from spontaneously hypertensive (SH) rats of the Okamoto strain. The total concentration of aorta glycosaminoglycans in the SH rat was 33% higher than in the WKY rat, due to a 164% increase in chondroitin 4- and 6-sulfate. The content of dermatan sulfate (DS), hyaluronic acid (HA), and heparan sulfate (HS) was similar in the two strains. The 4-wk-old SH rat also had an increase in chondroitin sulfate (CS) compared to the 4-wk-old WKY rat, without any change in DS, HA, or HS. The Wistar rat had approximately the same concentration of CS und DS in the aorta as the WKY rat, but HS und HA were reduced by 62 and 37%, respectively. The galactosaminoglycans (CS and DS) were heterogeneous on cellulose acetate electrophoresis and exhibited a different pattern for each of the three strains. Undersulfated CS accounted for 15% of the total CS in WKY aorta but was present in only trace amounts in the SH aorta; 2% of the CS from the Wistar aorta was undersulfated. In all three strains, DS was exclusively 4-sulfated, and the CS contained approximately equal amounts of 4- and 6-sulfated galactosamine residues. Ultrastructural studies demonstrated that the HS was localized in the subendothelial matrix and the pericellular region surrounding the medial smooth muscle cells. CS and DS were primarily associated with collagen in the media. In the SH rat aorta the subendothelial matrix was thicker, and there was a relative increase in the CS/DS in the smooth muscle cell pericellular matrix. We suggest that, if similar alterations in CS proteoglycans are present in the resistance vessels, these changes may contribute to the increased peripheral vascular resistance in the hypertensive animal.
Animals↗
Proteoglycans and hypertension. II. [35S]sulfate incorporation into aorta proteoglycans of spontaneously hypertensive rats.
Spontaneously hypertensive (SH) rats are known to have an increased content of chondroitin sulfate (CS) proteoglycans (PG) in the aorta as compared to normotensive Wistar Kyoto (WKY) rats. In the present study we have compared WKY and SH rat aortas with respect to [35S]sulfate incorporation in vivo and in vitro. The specific activity (cpm/mg aorta) of the total glycosaminoglycan (GAG) pool from SH rat aorta, measured 48 h after intraperitoneal injection of [35S]sulfate, was twice as high as that of WKY aorta GAG. After in vitro incubation of aortas for 4 or 6 h, the specific activity (cpm/mg aorta) of glycosaminoglycans from SH rat was 2.4- to 7.1-fold higher than in controls. Labeled PG were extracted with 4 M guanidine from aortas which had been incubated with [35S]sulfate, and chromatography of the extract on Sepharose CL-6B yielded two incompletely resolved peaks, one emerging with the void volume (peak I) and one in a more retarded position (peak II). Peak I (WKY) contained nearly equal amounts of CS and HS (53 and 46%, respectively) and a small amount of DS (8%). Peak II (WKY) (Kav, 0.34) was divided into two fractions; the fraction of larger molecular weight (II A) contained 43% CS, 35% DS, and 20% HS, whereas the smaller fraction (II B) contained 40% CS, 51% DS, and 5% HS. In each corresponding pool from SH rat aorta, a similar proportion of HS was found, but the DS content was approximately half, and the CS content was correspondingly greater. The estimated molecular weights of the CS/DS chains in peaks I, II A, and II B from WKY aorta were 34,600, 18,800, and 11,600 daltons, respectively, whereas the corresponding values for the SH rat aorta pools were 32,300, 24,700, and 17,000 daltons, respectively. The proportions of 4- and 6-sulfated galactosamine residues as well as the degree of sulfation of the CS/DS PG were similar in the two strains. The HS-PG was larger in the WKY rat aorta and was made up of larger HS chains (Mr 26,600 vs. 16,100); however, the degree of sulfation was apparently similar in the two strains. These results suggest that the rates of PG synthesis and/or degradation and the PG structure are altered in the SH rat aorta.
Animals↗